Mutations in the human BMP4 and BMP2 genes have been associated with osteoporosis and these BMPs signal through a variety of BMP receptors. The role for BMP2 and BMP4 in bone biology is limited since deletions of BMP4 and BMP2 are embryonic lethals. Data from our lab has shown that conditional knock-out of BMP4 in matrix producing osteoblasts, using Cre/loxP, results in postnatal animals with an osteopenic phenotype. BMP4 cKO mice have decreased osteoblast activity and increased osteoclasts in older animals. We have recently shown that the Shh responsive gene, Gli2, directly regulates BMP2 expression and transcription in osteoblast precursors through Gli response elements. Our hypothesis is that BMP4 and BMP2 are required at early mesenchymal precursor cells to drive the precursors to a commitment osteoblast stage before expansion and differentiation to mature osteoblasts. Further elevated BMP levels and signaling through BMP receptor 1A, is then required to drive the development of the mature matrix/mineralizing osteoblasts and osteocytes. We will test this hypothesis by selectively removing BMP2 and BMP4 at two different osteoblast stages, the early preosteoblast stage and at later matrix producing osteoblast stage. We will then attempt to rescue some of these bone phenotypes by selectively activating one of the important BMP receptors in bone, BMPR1A. Specific Aim 1 will be directed at determining the specific and overlapping roles of BMP4 and BMP2 in osteoblast biology, using the Osterix-CreERt2 mouse model to temporally delete BMP4 and/or BMP2 in preosteoblast stage, specifically in animals after birth. Specific Aim 2 will be to determine the role of BMP2 and BMP4 in later stage osteoblasts, using the 3.2 Col1a1-CreERtm model to induce deletions after birth. Specific Aim 3 will be to determine the mechanism of action of BMP2 and BMP4 in vitro, using bone marrow mesenchymal precursor cultures in combination with BMP2 and BMP4 deletions in vitro with Adenovirus Cre. Growth, apoptosis, differentiation, and altered pathways will be determined, as well as gene expression patterns using microarray analysis. Specific Aim 4 will be directed at determining what aspects of the bone specific deletions of BMP2 and BMP4 are due to signaling through the BMP receptor 1A. This hypothesis will be tested using quantitative in situ hybridization, immunocytochemistry. Western analysis of altered signaling pathways will also be determined in a quantitative manner. b-catenin/TCF and BMP signaling reporter mice and lineage marker mice will be used to determine levels of these pathways and alterations in the BMP2 and BMP4 deletion mice. In vitro primary osteoblast cell cultures, before and after specific gene deletion will be used to determine candidate mechanism of action of the single BMP2 or BMP4 and the combined deletion of both BMP2 and BMP4.